We have shown that T5 phage codes for the synthesis of 24 different tRNA species. The tRNA genes have been mapped and are located in six clusters in the C segment of T5 DNA. Using DNA fragments generated by a series of restriction endonucleases, we are presently in the process of determining the nucleotide sequence of those DNA fragments which contain the T5 tRNA genes. A second project concerns the regulation of tRNA synthesis in mammalian tissues. We have been able to separate a wide variety of tRNA species from crude preparations of rat tissue by two-dimensional gel electrophoresis. The tRNAs are prelabeled by injection of rats with 32Pi. The labeled tRNAs are located on the gels by autoradiography, extracted, and identified by charging with 3H-amino acids. Examination of tRNA gel-profiles from liver and kidney tissues show that they are not identical. Differences in gel-profiles may be due to differences in tRNA modification enzymes in the respective tissues or to differences in gene expression. We hope to examine tRNA synthesis in various tissues derived from animals under different stages of development, in cells following virus infection, and after the administration of chemical carcinogens.